This project is focused on understanding clathrin-independent forms of endocytosis (CIE). Endocytosis that occurs without clathrin coats occurs in all cells but is poorly understood. We are interested in studying the cargo proteins that enter cells by this mechanism, their intracellular itinerary once they have been internalized and whether they contain amino acid sequences that allow for specialized sorting within cells. We have been identifying new cargo proteins and found that a subset of these proteins take alternative traffic routes once they have entered cells. The major histocompatibility complex Class I protein (MHCI), is a prototypical clathrin-indepenent cargo protein and after internalization it reaches endosomes that contain cargo proteins such as the transferrin receptor that enter via clathrin-depenent endocytosis. From there, MHCI travels either to late endosomes and lysosomes where it is degraded or on to recycling tubules that return MHCI back to the cell surface. CD44, CD98, and CD147, however, show an altered itinerary in many cells where they traffic directly into the recycling tubules and avoid trafficking to lysosomes. Consistent with this altered itinerary, CD44, CD98 and CD147 are long-lived proteins and are not degraded like MHCI, which is routed to lysosomes. We are interested in the role that ubiquitin modification of cargo proteins plays in regulating the sorting of cargo and routing of cargo to lysosomes for degradation. We previously showed that over expression of the MARCH8 E3 ubiquitin ligase leads to ubiquitination and degradation of CD98, normally a long-lived protein (Eyster et al 2011). We also published that expression of a de-ubiquitinase (DUB) or ubiquitin-specific protease (USP) can counteract the effect of expression of MARCH8. This DUB is called TRE17/USP6 and was previously shown to be unregulated in ABC bone cancer. Expression of TRE17 specifically de-ubiquitinates CD98 and returns its trafficking to the normal pathway (i.e. avoidance of lysosomes) (Funakoshi et al, 2014). However TRE17 did not rescue the effect of MARCH8 on degradation of the transferrin receptor. Interestingly, our collaborator was examining the effect of cytomegalovirus infection on trafficking of CIE cargo proteins. With a closer look, we found that CIE cargo proteins (including CD147) were trapped in sorting endosomes in CMV-infected cells. Expression of the ubiquitin-specific protease USP6 (or TRE17) rescued this phenotype allowing CD147 to return to the cell surface. Furthermore the effect of TRE17 expression was dependent upon the de-ubiquitinase activity of TRE17/USP6 (Zeltzer et al 2018). This suggests that infection with CMV may either suppress the activity of USP6 or else induce ubiquitination of CD147. We are now analyzing the results of an siRNA screen of human de-ubiquitinases (DUBs), knocking down the 100 DUBs in the human genome to identify those DUBs that are responsible for protecting CD98 and CD147 from lysosomal degradation. We obtained several hits and are currently validating these hit and following up on them with further studies. One of the hits was in fact USP6/TRE17, which was re-assuring and a kind of positive control since we had demonstrated that overexpression of TRE17 could de-ubiquitinate CD98 and protect it from degradation (Funakoshi et al 2014). Loss of USP6/TRE17 increases the turnover of both CD98 and CD147 and leads to their altered trafficking to lysosomes. However it did not affect the turnover of MHC Class I.